The goal of the proposed research is to correlate the structure-function relationship in proteins using hemoglobin (Hb) as a model. There are several features inherent in Hb which make this molecule a unique choice for gaining insights into the signal pathway for information transfer from one subunit to another at the atomic level. In order to achieve our goal, we plan to apply the techniques of biochemistry, biophysics, molecular biology, and structural biology to our research and to correlate the results obtained from nuclear magnetic resonance spectroscopy, x-ray crystallography, time-resolved fluorescence spectroscopy, resonance Raman spectroscopy, computer modeling, and equilibrium and kinetic studies of binding of ligands to novel Hbs created in our laboratory. Some of these experiments will be carried out in collaboration with leading experts. The proposed research is a comprehensive biophysical and molecular biological investigation of Hb and consists of three parts: (i) development of improved Hb expression system(s) and procedures to improve the yield of various recombinant Hbs needed for research; (ii) new approaches to investigate the molecular basis for the cooperativity and oxygen affinity of Hb; and (iii) new approaches to investigate the molecular basis for the polymerization of sickle cell Hb (Hb S). An improved expression system in Escherichia coli and better isolation and purification protocols will allow us to produce various Hbs needed for our research more efficiently. A better understanding of the various structures of Hb that exist in crystalline and solution states will allow us to gain new insights into the nature of the subunit interactions and of the environment of the heme pockets that give rise to the cooperative oxygenation process and oxygen affinity of Hb. This kind of information will be very useful in designing Hbs with desired degree of cooperativity and of oxygen affinity. A possible application of this research is in the area of Hb-based blood substitutes. A better understanding of the intermolecular contacts in the Hb S polymer will provide new ideas for the development more effective anti-sickling drugs and gene therapy for patients with sickle cell anemia. This is an opportune time to carry out a comprehensive research on Hb because the traditional thinking regarding the structure-function relationship in this protein molecule has been challenged by the recent results published in the literature. Furthermore, a convenient expression system for producing any desired Hbs in E. coli as well as powerful modern structural biology techniques are available to the proposed research. Thus, the results to be obtained from the proposed research not only will provide new understanding relating to the structure-function relationship in Hb as well as new insights into multimeric-protein interactions, allosteric regulation, molecular recognition, and protein engineering in general, but also can lead to advances in clinical medicine such as gene therapy for patients with hemoglobinopathies and the development of effective Hb-based blood substitutes. Thus, hemoglobin research is an excellent illustration of how the discoveries from basic research in proteins can make important contributions to medicine and biotechnology.